Unravelling the role of NAADP/TPC2 Ca2+ signalling during angiogenesis and tumor progression

Two pore channels (TPCs) represent an emerging class of ion channels principally involved in calcium (Ca2+) mobilization elicited by nicotinic acid adenine dinucleotide phosphate (NAADP) [1, 2]. TPCs localize in the endo-lysosomal compartments and they exist in three isoforms. In particular, we have previously demonstrated the pivotal role of NAADP/TPC2 pathway in angiogenesis and mouse melanoma [3, 4]. In this study, we have explored and characterized the potentiality of Naringenin (Nar), a natural flavonoid, with the aim to investigate a novel pharmacological tool able to interfere with TPC2 activity both in endothelium and in B16 melanoma cells. We demonstrated that Nar impairs intracellular Ca2+ responses of endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (known to elicit NAADP-independent responses). In addition, we have observed that Nar is able to inhibit angiogenesis in vitro and in an established in vivo murine model. Taken as a whole the present data suggest that Nar inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular Ca2+ signalling. Several experimental evidence have shown that Ca2+ is involved in fundamental cancer cell activities such as proliferation, migration, invasiveness, metastasis dissemination and survival of tumor cells [5]. Based on this evidence we have focused our attention on the study of VEGF-dependent Ca2+ signalling in melanoma cancer cells [4]. In this work, we also demonstrated that Nar inhibits VEGF and NAADP-dependent Ca2+ release also in B16 melanoma cells. Moreover, Nar impairment of B16 migration/ proliferation and vasculogenic mimicry strengthens the hypothesis that TPC2 represent a potential target for control of the progression of melanoma and possibly other tumors. References: 1. Calcraft, P.J., et al., NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature, 2009. 459(7246): p. 596-600. 2. Pitt, S.J., B. Reilly-O'Donnell, and R. Sitsapesan, Exploring the biophysical evidence that mammalian two-pore channels are NAADP-activated calcium-permeable channels. J Physiol, 2016. 594(15): p. 4171-9. 3. Favia, A., et al., VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2- dependent Ca2+ signaling. Proc Natl Acad Sci U S A, 2014. 111(44): p. E4706-15. 4. Favia, A., et al., NAADP-Dependent Ca(2+) Signaling Controls Melanoma Progression, Metastatic Dissemination and Neoangiogenesis. Sci Rep, 2016. 6: p. 18925. 5. Monteith, G.R., N. Prevarskaya, and S.J. Roberts-Thomson, The calcium-cancer signalling nexus. Nat Rev Cancer, 2017. 17(6): p. 367-380